Phosphoinositide 3-kinase (PI3K) has been implicated in b2-adrenergic receptor (b2-AR)/Gi-mediated compartmentation of the concurrent Gs-adenylyl cyclase-cAMP-PKA signaling, negating b2-AR-induced phospholamban phosphorylation and the positive inotropic and lusitropic responses in cardiomyocytes. However, it is unclear whether PI3K is involved in the signal transduction of b1-AR, the predominant b-AR subtype expressed in cardiomyocytes. Here, we show that selective b1-AR stimulation by norepinephrine in the presence of a1-AR and b2-AR blockade markedly increases PI3K activity in adult rat ventricular myocytes. Inhibition of PI3K by LY294002 (5.10-6 mol/L) significantly enhances b1-AR-induced increases in L-type Ca2+ currents, intracellular Ca2+ transients, and myocyte contractility, without altering the receptor-mediated phosphorylation of phospholamban, a key cardiac PKA target protein. The potentiating effects of LY294002 are completely prevented by inhibiting Gbg signaling with a peptide inhibitor, bARK-ct, but not by disrupting Gi function with pertussis toxin, suggesting that the Gbg heterodimer dissociated from Gs plays an essential role in b1-AR-activated PI3K signaling. Moreover, direct stimulation of adenylyl cyclase by forskolin also elevates PI3K activity, and inhibition of PI3K enhances the contractile response to forskolin-induced contractile response or activation of PKA with a cAMP analogue, CPT-cAMP. In contrast, inhibition of PI3K affects neither the basal contractility nor high extracellular Ca2+-induced increase in myocyte contraction. These results suggest that both the Gsa-stimulated cAMP signaling and the free Gbg released from Gs proteins are likely involved in b1-AR-mediated PI3K activation, which, in turn, negates b1-AR-induced positive inotropic effect via inhibiting PKA-dependent sarcolemmal Ca2+ influx and the subsequent increase in intracellular Ca2+ transients, without altering the receptor-mediated phospholamban phosphorylation, in intact cardiomyocytes.